The ability to detect exceedingly small amounts of a nucleic acid in a sample generally requires the amplification of the amount of the target nucleic acid. This is especially important for the detection of human retroviruses, where positive samples may contain only 5-10 target molecules in 10.sup.6 cells.
The preferred method for amplifying target DNA has been the polymerase chain reaction (PCR) technique. The technique has been described in U.S. Pat. No. 4,683,195, issued Jul. 28, 1987 to Mullis, et al., U.S. Pat. No. 4,683,202, issued Jul. 28, 1987 to Mullis, U.S. Pat. No. 4,800,159, issued Jan. 24, 1989 to Mullis, et al., U.S. Pat. No. 4,889,818, issued Dec. 26, 1989 to Gelfand, et al., and U.S. Pat. No. 4,902,624, issued Feb. 20, 1990 to Columbus, et al., all of which are incorporated herein by reference.
In general, the PCR reaction involves the use of a pair of specific oligonucleotide primers to initiate DNA synthesis on a target DNA template. Two oligonucleotide primers are used for each double-stranded sequence to be amplified. The target sequence is denatured into its complementary strands. Each of the primers, which are sufficiently complementary to a portion of each strand of the target sequence to hybridize with it, anneals to one of the strands. The primers are extended, using nucleosides in the sample and a polymerization agent, such as heat-stable Taq DNA polymerase. This results in the formation of complementary primer extension products, which are hybridized to the complementary strands of the target sequence. The primer extension products are then separated from the template strands, and the process is repeated until the desired level of amplification is obtained. In subsequent cycles, the primer extension products serve as new templates for synthesizing the desired nucleic acid sequence.
By repeating the cycles of denaturation, annealing, and extension, the original target DNA can be amplified exponentially according to the formula 2.sup.n, where n is the number of cycles. In theory, 25 cycles, for example, would result in a 3.4.times.10.sup.7 -fold amplification. However, since the efficiency of each cycle is less than 100%, the actual amplification after 25 cycles is about 1-3.times.10.sup.6 -fold. The size of the amplified region is generally about 100-400 base pairs, although stretches of up to 2 kb can be amplified. See Keller and Manak, DNA probes (New York: Stockton Press, 1989), pgs. 215-216.
The three basic steps of the PCR reaction--denaturation, annealing, and extension--are driven and controlled by the temperature of the reaction mixture, with each step occurring at a different temperature. Somewhat different temperature ranges are disclosed for each of the three steps in the above-referenced patents. However, as time passed, those skilled in the art have settled on fairly standard temperatures for each of the steps in the cycle. Thus, Gelfand, et al., discloses a denaturing temperature range of about 90.degree.-105.degree. C., preferably 90.degree.-100.degree. C., an annealing temperature range of about 35.degree.-65.degree. C., preferably 37.degree.-60.degree. C., and an extension temperature range of about 40.degree.-80.degree. C., preferably 50.degree.-75.degree. C. For Taq polymerase, which is the overwhelmingly preferred polymerase for the PCR reaction, Gelfand, et al., refers to an annealing temperature range of about 45.degree.-58.degree. C. and an extension temperature range of about 65.degree.-75.degree. C. Columbus, et al., which is directed to a temperature cycling cuvette for use in PCR, refers to temperature ranges of 92.degree.-95.degree. C. for the denaturing step, 50.degree.-60.degree. C. for the annealing step, 70.degree. C. for the extension step. Keller and Manak, cited above, refer to a denaturation temperature of about 93.degree. C., an annealing temperature of 37.degree.-55.degree. C., and a primer extension temperature of 70.degree. C.
The PCR technique has been modified to permit the amplification of viral RNA. See Murakawa, et al., DNA, 7:287-295 (1988), which is incorporated herein by reference. The article discloses the amplification of sequences from HIV-1 RNA templates for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and ARC patients. Total nucleic acid is isolated from infected cells, and the DNA is digested with RNase-free DNase so that it does not contribute to the final PCR product. A cDNA copy of a target sequence of the viral RNA is synthesized, using the PCR primers and reverse transcriptase. The one primer complementary to the RNA serves to initiate cDNA synthesis.
Several different formats have been used for the detection of PCR products. Generally, a radioactive or nonradioactive labeled probe that is complementary to the target sequence is used. The hybridization of the probes to the amplified target sequence, and the subsequent detection of the labeled moiety results in the detection of the target sequence. Nonradioactive labeled probes are generally more desirable because they obviate the need for special handling procedures. However, they may not generate as intense a signal, or the signal may be obscured by background "noise." Thus, there is a need for enhancing the intensity of the signal in such probes.
The PCR technique is a revolutionary one, and it is widely used. Nevertheless, it does have significant drawbacks. The most serious of these is nonspecific hybridization, which results in false positives. Avoiding nonspecific hybridization requires ultrapure reagents. Unfortunately, for most clinical and diagnostic applications, it is desirable to use "dirty" samples, which presents a major problem, unless time-consuming and expensive sample preparation is undertaken.
Another important drawback to the PCR technique is the time involved in amplification. Although the usual six hour time period for PCR is far superior to an alternative technique such as cloning, which can take days or weeks, it would still be desirable to cut amplification time by one-half to two-thirds.
The present invention overcomes these drawbacks of the PCR technique, and it provides an improved detection system. The invention provides methods for the rapid amplification and detection of nucleic acids in which the denaturing, annealing, and extension steps occur all at the same temperature or, alternatively, at only two different temperatures, thus providing for faster cycling. In addition, the invention provides amplification methods where the annealing temperature is higher than the prior art temperatures, thus eliminating nonspecific hybridization. Finally, the invention provides improved methods of detecting the amplified nucleic acids through a two-stage signal amplification.